The dissemination of multidrug-resistant and hypervirulent Klebsiella pneumoniae clones across the Kingdom of Saudi Arabia

Abstract

Klebsiella pneumoniae is a Gram-negative bacterium associated with a wide range of community- and hospital-acquired infections. The emergence of clonal hypervirulent strains resistant to last-resort antimicrobial agents has become a global concern. The Kingdom of Saudi Arabia (KSA), with its diverse population and high tourism traffic, serves as a platform where the spread of multidrug-resistant (MDR) strains are facilitated. However, the knowledge of epidemiology and population diversity of MDR K. pneumoniae in KSA is scarce. We conducted a comprehensive genomic survey on 352 MDR K. pneumoniae isolates systematically collected from bloodstream and urinary tract infections in 34 hospitals across 15 major cities in KSA during 2022 and 2023. Whole-genome sequencing on the isolates was performed, followed by genomic epidemiology and phylodynamic analysis. Our study revealed a dynamic population characterized by the rapid expansion of several dominant clones, including, ST2096, ST147, and ST231, which were estimated to have emerged within the past decade. These clones exhibited widespread dissemination across hospitals and were genetically linked to global strains, particularly from the Middle East and South Asia. All major clones harboured plasmid-borne ESBLs and carbapenemase genes, with plasmidome analysis identifying multiple IncH, IncA/C and IncL plasmids underlying the MDR-hypervirulent phenotype. These plasmids were shared between major clones and became acquired on the same time scales as the expansion of the dominant clones. Our results report ST2096 as an emerging MDR-hypervirulent clone, emphasizing the need for monitoring of the circulating clones and their plasmid content in the KSA and broader West Asia.

Keywords: Klebsiella pneumoniae; antimicrobial resistance; hypervirulence and multidrug resistance (MDR); precision epidemiology; whole genome sequencing.

Figure 1.

The distribution of K. pneumoniae clones in the study cohort. (A) The frequency of STs across provinces. The size of the pie charts corresponds to the number of isolates. (B) Proportion of samples originating from different sources of isolation. The terms “BC” and “UC” stand for blood and urine culture, respectively. (C) SNP diversity of isolates in each province. Diversity is measured as the pairwise SNP distance between isolates originating from the same province. The colours represent the regions in the country.

Figure 2.

Phylogenetic tree K. pneumoniae in the KSA. The tree included 361 K. pneumoniae genomes from the current study and 328 genomes from a single-hospital study in Jeddah [18]. The tree is an approximate maximum-likelihood phylogenetic tree from the alignment of 196,300 SNPs. Strains for STs with more than ten representative strains are shown. The resistance and virulence scores were computed with Kleborate. The dotted lines show the cut-off used for defining multidrug resistance and virulence factor genes. The plasmid bands correspond to the mapping coverage for the plasmids containing carbapenemase and ESBLs from long-read sequencing. “BC” and “UC” correspond to blood and urine culture samples, respectively.

Figure 3.

Population dynamics of major clones. (A) The estimated population dynamics parameters from the phytogeography analysis. Clock rates show the rates based on the number of substitutions per site per year. The migration rates show the parameters for all the cities and the rates. The unit for migration is the number of migration events per year. The most recent common ancestor (MRCA) age indicates the time at which the clone formed. The error bars show the 95% confidence intervals. (B) The skyline growth plot. The shaded plot shows the 95% confidence intervals. (C) The recent spread of ST147, ST231 and ST2096 clones across the country. The trajectories show the inferred dissemination (migration routes) across the country. The circle sizes and colour intensity and rate show the relative marginal migration rate per city.

Figure 4.

Mixing of Saudi clones with the global collection. (A) The SNP clusters (clusters of genomes fewer than 50 SNPs apart) as derived from the pathogen detection database (see Methods for the definition of the clusters). (B) Neighbor-joining tree for the clusters that contained more than two isolates from Saudi Arabia and the global collection in the database. The colour tips represent strains from Saudi Arabia and other regions.

Figure 5.

Frequency of clinically significant resistance and virulence genes across major clones of K. pneumoniae from blood stream infections in the KSA. (A) The frequency of resistance genes across the major STs. The “***” symbol indicates the significance level of the higher number of resistance genes from the Wilcoxon rank-sum test (p-value < 0.001). (B) ESBL, colistin resistance and carbapenemase genes across STs. The numbers show the resistance scores (see methods for more information). (C) The distribution of aerobactin (iuc) and yersiniabactin (ybt) across major clones (STs) and other STs. The numbers show the virulence scores (see methods for more information). (D) The integration of the virulence score and resistance score for the STs in the population. The shaded areas define the hypervirulent, multidrug resistant and hypervirulent-MDR resistant areas. The scores and definitions of hypervirulence and MDR were adopted from Kleborate, and details are provided in Supplemental Table S1. The x- and y-axes correspond to the average virulence and resistance scores for isolates within each ST, respectively.

Figure 6.

The retrieved plasmid fragments that contained a resistance gene. The top panel shows the length of the plasmid in base pairs. The bubble plot shows the frequency of plasmids across major clones. We mapped the short reads to the plasmid fragments and reported presence of plasmid if the mapping coverage was above 90%. The shaded boxes show the STs of the isolate from which the plasmid fragment was retrieved. The pIncH1, pOXA232 and pOXA48 fragments were from the ST2096 isolates, previously studied [18].